Platelet transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for platelet transfusions is often limited by shortage in the platelets (PTL) supply due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. It is therefore critical to create an efficient, donor-independent source of PTLs that could complement-and possibly replace-the current donor- dependent transfusion system. The search for a different source of platelets for transfusion was accelerated by the recent advances in hematopoietic stem cell expansion which became a promising source for generating platelets ex vivo. However, creating a clinically relevant platelet product ex vivo has been hampered by technological and quantitative limitations. The major barrier in generating PTLs suitable for transfusion is the low number of PTLs that can be produced by cultured megakaryocytes (MK) ex vivo and the difficulties in recapitulating the complexity of physiological human thrombopoiesis in culture. The studies proposed in this application are designed to develop an alternative approach by creating a MK cell product that can generate platelets in vivo after its transfusion into patients. Creating such product will not only circumvent the technological hurdles and costs associated with ex vivo PTL production but also provide the foundation for developing a cell-based therapy for thrombocytopenic patients and possibly preclude alloimmunization. In addition, MKs generated from this study will be advanced to a manufacturing phase to create the first commercially available source of CD41+ MKs for use in research. A commercial supply of MKs would dramatically accelerate the pace of research in human thrombopoiesis. This goal will be accomplished by implementing a new culture system protocol in which a MK cell product comprising MK progenitors/precursors, immature MKs, and mature MKs, will be generated from a validated source of cord blood-derived CD34+ hematopoietic stem cells expanded by means of epigenetic reprograming by treatment with valproic acid. These proposed feasibility studies are designed to (1) characterize the resultant MKs phenotypically and functionally, (2) determine the MKs' potency ex vivo and in vivo in transplantation assays in an immunodeficient mouse model, and (3) test the MKs' ability to remain viable and functional following cryopreservation and storage. These studies will lead to development of a platform for developing a commercial MK cell product for clinical use to treat thrombocytopenia and for laboratory research.